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Seeding and Cross-Seeding Aggregations of A#40 and its N-Terminal Truncated Peptide A#11-40 Xiuping Hao, Jie Zheng, Yan Sun, and Xiaoyan Dong Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03599 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019
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Seeding and Cross-Seeding Aggregations of Aβ40 and its N-Terminal
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Truncated Peptide Aβ11-40
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Xiuping Hao,† Jie Zheng,ǂ Yan Sun,† and Xiaoyan Dong,*,†
5 6
† Department
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the Ministry of Education, School of Chemical Engineering and Technology, Tianjin
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University, Tianjin 300072, China
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ǂ
10
of Biochemical Engineering and Key Laboratory of Systems Bioengineering of
Department of Chemical and Biomolecular Engineering, The University of Akron, Akron,
Ohio 44325, United States
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ABSTRACT:
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In the amyloid plaques of Alzheimer’s disease (AD) patients, a large number of N-terminal
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truncated amyloid β (Aβ) peptides such as Aβ11-40 have been identified in addition to the full
4
length Aβ peptides. However, little is known about the roles of the N-terminal truncated
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peptides in AD pathological process. Herein, seeding and cross-seeding aggregations of Aβ40
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and its N-terminal truncated Aβ11-40 were investigated in solution and on the surfaces of chips
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with immobilized seeds by extensive biophysical and biological analyses. The results showed
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that Aβ40 and Aβ11-40 aggregates could seed both homologous and heterologous aggregations
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of the two monomers. However, the capability and characteristics of the seeding
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(homologous aggregation) and cross-seeding (heterologous aggregation) were significantly
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different. Aβ40 seeds showed stronger acceleration effects on the aggregations than Aβ11-40
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seeds and induced β-sheet-rich fibrous aggregates of similar cytotoxicities for the two
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monomers. This indicates that Aβ40 and Aβ11-40 had similar aggregation pathways in the
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seeding and cross-seeding on Aβ40 seeds. By contrast, Aβ11-40 seeds led to different
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aggregation pathways of Aβ40 and Aβ11-40. Pure Aβ11-40 aggregates had higher toxicity than
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Aβ40 aggregates, and as seeds, Aβ11-40 seeds induced Aβ40 to form aggregates of higher
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cytotoxicity. However, homologous Aβ11-40 aggregates induced by Aβ11-40 seeds showed
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lower cytotoxicity than pure Aβ11-40 aggregates. The results suggest that Aβ11-40 plays an
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important role in the pathological process of AD.
20 21
KEYWORDS: Amyloid β-peptide; N-terminal truncated peptide; seeding; cross-seeding;
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surface aggregation.
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INTRODUCTION Alzheimer’s disease (AD) is an age-related neurodegenerative disease characterized by
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progressive
cognitive
impairment,
disorientation,
memory
impairment
and
other
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symptoms.1,2 Over 40 million people in the world are affected by AD, and this figure is
5
expected to double in the next few decades.3 The two pathological hallmarks of AD are
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intracellular neurofibrillary tangles formed by tau proteins and extracellular senile plagues
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formed by amyloid β-peptides (Aβ).4,5 Over the years, many investigations have provided
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support for a central role of Aβ fibrillogenesis in AD pathogenesis.6
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Aβ is derived from proteolytic cleavage of the larger amyloid-β protein precursor (APP).7
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The 40 or 42 amino acid peptides Aβ1–40/42 are generated through the action of β- and γ-
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secretase complex, and they can aggregate into highly structured, cytotoxic aggregates in
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vivo.8 However both the β- and γ-secretases have variable site specificities for APP and thus
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generate Aβs of different lengths.9 In addition, some other proteases, such as
14
metalloproteinase-9 (MMP9),10 aminopeptidases,11 caspase12 or neprilysin,13 possibly involve
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in N-truncated Aβ generation. Several N-terminally truncated Aβ isoforms, including Aβ2–x,
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AβpE3–x, Aβ4–x, and Aβ11–x have been reported to be more prone to aggregation and more
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toxic than non-truncated Aβ peptides.14-21 Cleavage by β-secretase at Glu11 (β' site) produces
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an N-terminally truncated peptide Aβ11-40/42.22 It has been shown that the amount of Aβ11-40/42
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in cerebrospinal fluid is comparable to that of Aβ1-42, and Aβ11-40/42 constitutes one-fifth of
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the senile plaque load.23 Notably, Aβ11-40/42 and N-terminal pyroglutamate forms are centered
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at the core of plaques in AD brains.24 In vitro studies have shown that Aβ11-40 is more prone
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to aggregation than Aβ40,25 and the addition of Aβ11-40 seeds can promote the formation of
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Aβ40 fibrils.14 Therefore, the N-terminal truncated peptide Aβ11-40 may play an important role
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in the pathological process of AD.
25
The aggregation process of amyloid in solution contains at least two microscopic steps:
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primary nucleation of the monomers and elongation of the fibrils. During the initial
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nucleation process, monomers bind into small oligomers that can reach sizes at which they
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grow faster by recruiting monomer compared to the rate they dissociate. The elongation of
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fibrils is the process that monomers bond to the ends of existing aggregates to lead the fast
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growth. In addition, secondary nucleation catalyzed by fibril surface may also play an
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important role. Secondary nucleation occurs in the presence of seeds which accelerate the
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formation of amyloid fibrils.26 The process of secondary nucleation can be homologous or
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heterologous. The association and co-aggregation of different amyloid proteins, a process
8
known as cross-seeding has been reported.27,28,29 Because most amyloid peptides have similar
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aggregation kinetics and structures, amyloid seeds may homologously or heterologously
10
catalyze the formation of amyloid fibrils.
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Using a home-built wireless quartz crystal microbalance (QCM) biosensor, Ogi and his co-
12
workers monitored the binding of Aβ40 and Aβ42 monomers to the Aβ40 and Aβ42 nuclei
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immobilized on the sensor surface for a long time.30,31,32 The structure formed in this
14
condition was significantly different from that formed in the solution under oscillating
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conditions, indicating that it’s important to study the interaction between immobilized nuclei
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and monomers. Some Aβ aggregates may be immobilized on the cell membrane or blood
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vessel wall in vivo. If Aβ monomers further associate on these aggregates, that may change
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the stability and permeability of the membrane, causing damage to the cell structure.33
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Therefore, it is of significance to study the aggregation of Aβ on the surface of immobilized
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nuclei.
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The quartz crystal microbalance with dissipation (QCM-D) is a sensor designed for mass
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and surface viscoelasticity detection. The working principle of QCM-D is mainly based on
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the piezoelectric characteristic of quartz crystals.34 When analyte absorbs to the immobilized
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molecules, the mass increase of the sensor would produce a frequency decrease, which
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allows the quantitative investigation of the binding kinetics. In addition, the dissipation
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change measured simultaneously can characterize the viscoelastic properties to provide
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useful structural information on the adsorbed materials.35 Now QCM-D has been used in the
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study of the growth process of amyloid fibrils and the effects of drug on fiber growth.31,36,37
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Among all the Aβ species, Aβ40 is the most abundant,38 and Aβ40 has an obvious
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aggregation lag phase, which is suitable for the analysis of seeding and cross-seeding
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aggregation kinetics and QCM-D measurement. Therefore, this work was designed to
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investigate the seeding and cross-seeding interactions between Aβ40 and Aβ11-40. We have
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studied their aggregation kinetics, morphology of the final aggregates, changes in secondary
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structure and toxicity of the aggregates to cells in the presence of Aβ40 or Aβ11-40 seeds using
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thioflavin T (ThT) fluorescence assay, atomic-force microscopy (AFM), far-UV circular
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dichroism (CD) and MTT assays. In addition, the deposition rates and structural changes of
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Aβ40 and Aβ11-40 monomers on immobilized homologous/heterologous seeds were studied by
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QCM-D. By elucidating the seeding and cross-seeding interactions between Aβ40 and Aβ11-40,
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our work has provided a clue for revealing the potential role of N-terminal truncated peptides
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in the pathogenesis of AD.
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MATERIALS AND METHODS
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Materials. Aβ40 and Aβ11-40 were obtained from GL Biochem (Shanghai, China).
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Hexafluoroisopropanol (HFIP), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
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bromide (MTT) and ThT were obtained from Sigma-Aldrich (St. Louis, MO, USA). 4-(2-
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Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was from Sangon Biotech
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(Shanghai, China). SH-SY5Y cells were from the Cell Bank of the Chinese Academy of
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Sciences (Shanghai, China). Dulbecco's Modified Eagle Medium/Ham's F-12 (DMEM/F12)
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and fetal bovine serum (FBS) were obtained from Gibco Invitrogen (Grand Island, NY). N-
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Hydroxysuccinimide
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hydrochloride (EDC) were obtained from J & K Scientific (Beijing, China). Bicinchonininc
(NHS)
and
1-ethyl-3-(3-dimethylaminopropyl)
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acid (BCA) kit was from Dingguo Biotech (Beijing, China). Other chemicals were from local
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sources with the highest purity. Deionized water was used for all solution preparations.
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Preparation of Aβ Monomers. Aβ was prepared as described in the literature.14, 39 Aβ40
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and Aβ11-40 were first dissolved in HFIP solutions to a concentration of 1.0 mg/mL. The
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solution was placed in quiescence at least 2 h and then sonicated for 30 min to destroy the
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pre-existing Aβ aggregates. HFIP was then removed using a vacuum freeze drier (Labconco,
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MO, USA). The lyophilized protein was stored in a refrigerator at −20 °C prior to use. The
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lyophilized protein was dissolved in 20 mmol/L NaOH and sonicated for 20 min. Then the
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solution was centrifuged at 16,000 g for 30 min at 4 °C. The upper 75% of the supernatant
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was carefully withdrawn and diluted with a HEPES buffer solution (30 mmol/L HEPES plus
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160 mmol/L NaCl, pH 7.4. The concentrations were determined by BCA kit (see below) and
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fixed to 25 μmol/L.
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Preparation of Aβ Seeds. Aβ40 or Aβ11-40 monomer solution was incubated for 48 h in an
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air bath of 37 °C with continuous shaking at 100 rpm, and then they were centrifuged at
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16,000 g for 30 min at 4 °C. The upper supernatant was withdrawn and the protein
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concentration was determined with the BCA kit to calculate the amount of Aβ40 and Aβ11-40
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aggregates in the precipitate by subtracting the Aβ amount in supernatant from that in Aβ
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monomer solutions, in which the Aβ concentration was 25 μmol/L. The precipitate was
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resuspended to a final concentration of 10 μmol/L (equivalent to monomer concentration) to
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obtain an Aβ seed suspension.
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BCA Assay for Protein Quantification. BCA assay was performed by following the kit
22
protocol. The prepared Aβ40, Aβ11-40 monomer solution, the upper supernatant of Aβ40 seeds
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and Aβ11-40 seeds were mixed with the BCA working reagent in 96-well plate. Different
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concentrations of the standard, bovine serum albumin (BSA) were also used for calibration.
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Specific volumes of different solutions added to each well are listed in Table S1. All samples
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were incubated at 60 °C for 30 min, then cooled to room temperature in 15 min. The
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Absorbance was measured at 562 nm in a Tecan Infinite M200 PRO (TECAN Austria
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GmbH, Grödig, Austria) plate reader. Three repeats were performed and the data averaged.
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The standard curve was drawn, and the protein concentration of samples were determined
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with the standard curve. The standard curve and its use are illustrated in Figure S1 and its
5
caption.
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Aggregation Kinetics Determined by in situ Thioflavin T Fluorescence. The samples
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of 200 μL were mixed in a 96-well plate, containing 25 μmol/L Aβ40 or Aβ11-40 monomers,
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Aβ40 or Aβ11-40 seeds (0.25, 0.5 or 0.75 μmol/L) and 25 μmol/L ThT in 30 mmol/L HEPES
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(pH 7.4). The plate was sealed to avoid evaporation of the sample and incubated in a Tecan
10
Infinite M200 PRO plate reader at 37 °C with 30 s shaking every 30 min. ThT fluorescence
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was monitored by excitation at 445 nm and emission at 480 nm. The Aβ samples containing
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no seeds were used as controls, and the samples containing only ThT and containing ThT and
13
Aβ seeds were used as blank controls. Each group of experiment contained three parallel
14
samples and the data were averaged after measurement. The standard deviation was reported
15
as an error bar in figures. The aggregation kinetic curves were fitted by the sigmoidal curves
16
using the following equation,40, 41
17
𝑦 = 𝑦0 +
𝑦𝑚𝑎𝑥 ― 𝑦0 1+𝑒
(1)
―(𝑡 ― 𝑡1/2)𝑘
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where y is the fluorescence intensity at time t, ymax and y0 are the maximum and minimum
19
fluorescence intensities, respectively, k is the apparent first-order aggregation constant, and
20
t1/2 is the time required to reach half the maximum fluorescence intensity. From the fitting
21
the lag phase time (Tlag) can be calculated from
22 23
2
(2)
𝑇𝑙𝑎𝑔 = 𝑡1/2 ― 𝑘
The average k and Tlag were obtained from three different repeats.
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Ex situ ThT Fluorescent Assay. Aβ40 and Aβ11-40 samples (25 μmol/L) without or with
25
Aβ40 and Aβ11-40 seeds (0.5 μmol/L) were incubated for 72 h by continuous shaking at 100
26
rpm and 37 °C. The final Aβ aggregates were observed using atomic force microscopy 7
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(AFM). For a ThT assay, 200 μL of the incubated sample was withdrawn carefully, and
2
mixed uniformly with 2 mL ThT solution (25 μmol/L ThT in 30 mmol/L HEPES, pH 7.4) in
3
a quartz cell. The ThT fluorescence emission at 480 nm were measured with excitation at 445
4
nm, 5 nm slit width on a fluorescence spectrometer (Perking Elmer LS-55, MA, USA) at
5
room temperature. The fluorescence intensities of the samples just containing seeds were
6
read and subtracted from the Aβ samples containing corresponding seeds. The fluorescence
7
intensity of the sample with HEPES was subtracted as background from each read with pure
8
Aβ. Three replicates were prepared for each sample and the data were averaged. The standard
9
deviation was reported as an error bar in figures.
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Atomic Force Microscopy. The morphologies of Aβ aggregates were observed by AFM
11
(CSPM5500, Benyuan, China). The samples were dropped on freshly cleaved mica substrates
12
for 5 min, carefully rinsed with deionized water, air-dried overnight at room temperature, and
13
then determined on AFM by at least three independent regions.
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Circular Dichroism (CD) Spectroscopy. The secondary structures of 25 μmol/L Aβ40
15
and Aβ11-40 aggregates formed in the absence and presence of 0.5 μmol/L seeds were
16
examined by CD spectroscopy. 300 μL of samples incubated for 72 h were placed into a
17
quartz cell of a 1 mm path length. The spectra were recorded between 260 and 190 nm in the
18
continuous scanning mode with a 1 nm bandwidth and a 100 nm/min scan rate using a J-810
19
circular dichroism spectropolarimeter (JASCO) at room temperature. All spectra were
20
corrected by subtracting the background and averaged by three successive scans for each
21
sample. Experimental ellipticity were converted into molar ellipticity by the following
22
equation,42
23
1000𝜃
(3)
[𝜃] = 𝑙 ∗ 𝑐 ∗ 𝑁𝑟
24
where θ is ellipticity (mdeg), [θ] is molar ellipticity (deg · cm2 · dmol-1), l is optical path
25
length (1 mm), c is concentration of Aβ peptides (2.5x10-2 mmol/L) and Nr is the number of
26
peptide residues (40 for Aβ40, 30 for Aβ11-40).
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Quartz Crystal Microbalance with Dissipation (QCM-D) Monitoring. Immobilization
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of Aβ fibrils onto the sensor surface were conducted as described in the literature.43 Gold-
3
coated AT-cut quartz crystals were treated with a piranha solution (98% H2SO4 : 30% H2O2
4
at 4:1) for 10 min, and then rinsed three times with deionized water. The electrodes were
5
immersed in 5 mmol/L 11-MUA/ultrapure ethanol solution for 48 h. After washing with
6
ethanol and water, the electrodes were activated for 30 min at room temperature using 0.4
7
mol/L EDC and 0.1 mol/L NHS. Then the electrodes were immersed in Aβ40 or Aβ11-40 seeds
8
solution for 12 h at room temperature. After washing with HEPES buffer and water, the
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remaining activated sites were blocked with 1 mol/L ethanolamine at pH 8.0 for 1 h. Six
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sensor chips were processed at the same time, and three of them were used to detect the
11
adsorption of homologous monomers to Aβ seeds, and the other three used for heterologous
12
monomers. The steps include: (1) place a chip in the QCM cell and rinse it with HEPES
13
buffer for a while until establishing a stable baseline; (2) pump 25 μmol/L Aβ monomer
14
solution over the sensor crystal surface for 30 min. The temperature of the QCM cell was
15
maintained at 37 ± 0.05 ℃, and a flow rate of 50 μL/min was maintained for all experiments.
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The resonance frequency and dissipation were recorded at the third, fifth, seventh, ninth and
17
eleventh overtone corresponding to 15, 25, 35, 45 and 55 MHz, respectively. Before and after
18
the experiments, the structure and morphology of the chip surface were observed by AFM.
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When the analyte is attached to the chip surface, the mass increases and the oscillation
20
frequency decreases accordingly, as expressed by the Sauerbrey equation,44
21
Δf = -aΔm
(4)
22
where Δf and Δm are frequency and mass change, respectively, and a is a scale factor. For
23
simple cases, such as a thin rigid attached layer in contact with air or a vacuum, the
24
Sauerbrey equation can be used to calculate mass changes.44 However, in the case of a
25
hydrophilic surface with sticky boundary conditions, for example, a chip surface with
26
amyloid fibrils, the mass change estimated by the Sauerbrey model may be four times
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higher.45 This is mainly due to the roughness of the fibril layer, which may capture water
2
molecules during the monitoring process. In this study, QTools software was used to fit
3
frequency change ΔF and dissipation change ΔD at the third, fifth, and seventh overtone and
4
to estimate the mass change Δm on the chip surface using the Kelvin-Voigt model,46 as
5
described by Eq.(5) and Eq.(6) in the following, 1
[
6
∆𝐹 ≈ ― 2𝜋𝜌0ℎ0
7
∆𝐷 ≈ 2𝜋𝜌0ℎ0
1
[
𝛿2 + ℎ1𝜌1𝜔 ― 2ℎ1
𝜂2
𝜂2 2
𝜂1𝜔2
𝛿2
+ 𝜔2𝜂21
()
𝜂2
𝜂2 2
()
𝛿2 + 2ℎ1
𝛿2
𝜇1𝜔
𝜇21
]
]
(5) (6)
𝜇21 + 𝜔2𝜂21
8
where 𝜌0 is the density of quartz plate, and ℎ0 its thickness; 𝜔 is the angular frequency; 𝜇1
9
the elastic shear modulus, 𝜂1 the shear viscosity, 𝜌1is the density and ℎ1is the thickness of the
10
overlayer, respectively, 𝛿2 the density and 𝜂2 the viscosity of bulk fluid. Frequency of
11
overtones 3, 5 and 7 along with dissipation were fitted pointwise using a least-squares
12
algorithm, varying the thickness (ℎ1), shear viscosity (𝜂1), and the shear modulus (𝜌1) of the
13
adsorbed layer. These fitted viscoelastic parameters were subsequently recombined to yield
14
the surface mass density Γ (ℎ1𝜌1). More details concerning Eq. (5) and Eq. (6) are available
15
from Voinova et al.46
16
Cell Viability Assay. The cytotoxicity of Aβ aggregates were assessed by MTT assay on
17
SH-SY5Y cells. A total of 8 × 103 SH-SY5Y cells/well were cultured in DMEM/F12
18
supplemented with 10% FBS, 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C
19
under 5% CO2 for 24 h in 96-well plates. Then, the cells were treated with 20 μL pre-aged
20
samples (Aβ40 or Aβ11-40 fibrils (25 μmol/L) incubated for 72 h without/with Aβ40 or Aβ11-40
21
seeds) and incubated for another 24 h. And then 10 μL MTT solution (5.5 mg/mL in HEPES)
22
was added into each well and incubated for 4 h. The medium was centrifuged at 1500 rpm for
23
10 min to remove the supernatant and replaced with 100 μL DMSO to dissolve the formazan
24
crystals, followed by shaking at 150 rpm for 10 min. Then, the absorbance at 570 nm was
25
measured in a multifunctional microplate reader (TECAN Austria GmbH, Grödig, Austria).
26
Six replicates were conducted and the data were averaged. The standard deviation was 10
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reported as an error bar in figures. The Tukey test was used to analyze the significance of
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each group of data.
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RESULTS AND DISCUSSION
4
Seeding and Cross-Seeding Aggregation Kinetics. To investigate seeding and cross-
5
seeding aggregation kinetics, namely, the effects of Aβ40 seeds or Aβ11-40 seeds on the
6
aggregation of Aβ40 and Aβ11-40, the aggregation kinetics were monitored by ThT
7
fluorescence. ThT fluorescence intensity of Aβ showed a sigmoidal appearance. The kinetic
8
growth curves obtained by fitting Eq. (1) to the experimental data are shown in Figure 1, and
9
the lag phase times and the apparent first-order aggregation constants obtained from Eq. (2)
10
are listed in Table 1. As shown in Figure 1 and Table 1, Aβ40 seeds and Aβ11-40 seeds not
11
only effectively promoted their own aggregation, shortened the lag phase time of Aβ40 and
12
Aβ11-40 significantly, but also promoted the cross-seeding between Aβ40 and Aβ11-40.
13
Especially when Aβ40 seeds were added into Aβ11-40 solution, the lag phase of Aβ11-40 was
14
eliminated (Figure 1B). This indicates that Aβ40 and Aβ11-40 seeds could accelerate the
15
homologous Aβ aggregation kinetics by secondary nucleation and heterologous Aβ
16
aggregation kinetics by cross-seeding. Then, Aβ40 and Aβ11-40 aggregation kinetics in the
17
presence of different concentrations of Aβ seeds were determined (Figure S2). It is seen that
18
the effect of seed concentration was not significant. Even at lower seed concentration (0.25
19
μmol/L), Aβ40 seeds could reduce the lag phase of Aβ11-40 longer than Aβ11-40 seeds did at
20
higher concentration (0.75 μmol/L). This indicates that Aβ40 seeds had high efficiency in
21
templating Aβ11-40 monomers.
22
In addition, the lag phase of Aβ11-40 was shorter than that of Aβ40 (Table 1), indicating that
23
Aβ11-40 aggregated faster in the nucleation phase under the experimental conditions, which is
24
consistent with that reported in literature.25 However, comparing the aggregation constant of
25
Aβ40 with that of Aβ11-40, it can be seen that the k value of Aβ40 alone was larger than that of
26
Aβ11-40, indicating that Aβ40 grew faster in the elongation phase. The addition of Aβ11-40 seeds
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decreased the k values of Aβ40 and Aβ11-40, indicating that Aβ11-40 seeds slowed down their
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elongation rates. When the plateau was reached, the fluorescence intensity of Aβ11-40 was
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lower than that of Aβ40, which suggests that Aβ11-40 aggregates had less β-sheet structure than
4
the Aβ40 aggregates. Meral and Urbanc 47 used the method of discrete molecular dynamic to
5
study the aggregation of N-terminal truncated peptides and also proved that Aβ11-40
6
aggregates contained less β-sheet.
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Morphology of Seeding-induced Aβ40 and Aβ11-40 Aggregates. First, the morphologies
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of pre-formed Aβ seeds were observed by AFM (Figure 2). Aβ40 seeds were fibrous in a few
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micrometers long (Figure 2A), while Aβ11-40 seeds were mainly spherical granules and
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irregular aggregates (Figure 2B). Barritt et al.14 observed that Aβ11-40 aggregates were short
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rods with length
